Process for the preparation of L-amino acids by attenuating the sucC and sucD genes

ABSTRACT

The invention relates to polynucleotides that contain polynucleotide sequences coding for the genes sucC and sucD, selected from the group  
     a) polynucleotide that is at least 70% identical to a polynucleotide coding for a polypeptide that contains the amino acid sequence of SEQ ID No. 2,  
     b) polynucleotide that is at least 70% identical to a polynucleotide coding for a polypeptide that contains the amino acid sequence of SEQ ID No. 3,  
     c) polynucleotide coding for a polypeptide that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 2,  
     d) polynucleotide coding for a polypeptide that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 3,  
     e) polynucleotide that is complementary to the polynucleotides of a), b), c) or d), and  
     f) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d) or e),  
     a process for the fermentative production of L-amino acids using coryneform bacteria in which the genes are present in attenuated form, and the use of the polynucleotide sequences as hybridization probes.

[0001] The present invention provides nucleotide sequences of coryneformbacteria coding for the genes sucC and sucD and a process for thefermentative production of amino acids, in particular L-lysine andL-glutamate, using bacteria in which the sucC- and/or sucD-gene is/areattenuated.

[0002] Prior Art

[0003] L-amino acids, in particular L-lysine and L-glutamate, are usedin human medicine and in the pharmaceutical industry, in the foodstuffsindustry, and most particularly in animal nutrition.

[0004] It is known that amino acids can be produced by fermentation ofstrains of coryneform bacteria, in particular Corynebacterium glutamicum(C. glutamicum). On account of the great importance of amino acidsefforts are constantly being made to improve the production processes.Improvements in production may involve fermentation technology measures,such as for example stirring and provision of oxygen, or the compositionof the nutrient media, such as for example the sugar concentrationduring fermentation or the working-up to the product form by, forexample, ion exchange chromatography, or the intrinsic output propertiesof the microorganism itself.

[0005] Methods involving mutagenesis, selection and choice of mutantsare used to improve the output properties. In this way strains areobtained that are resistant to antimetabolites or are auxotrophic forregulatorily important metabolites, and that produce amino acids.

[0006] For some years recombinant DNA technology methods have also beenused to improve Corynebacterium strains producing L-amino acids.

OBJECT OF THE INVENTION

[0007] The inventors have set themselves the task of providing newmeasures for improving the fermentative production of amino acids, inparticular L-lysine and L-glutamate.

DETAILED DESCRIPTION OF THE INVENTION

[0008] Where L-amino acids or amino acids are mentioned hereinafter, itis understood that these terms refer to one or more amino acids,including their salts, selected from the group comprising L-asparagine,L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine,L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.L-lysine and L-glutamate are particularly preferred.

[0009] The present invention provides an isolated polynucleotidecontaining a polynucleotide sequence selected from the group comprising

[0010] a) polynucleotide that is at least 70% identical to apolynucleotide coding for a polypeptide, that contains the amino acidsequence of SEQ ID No. 2,

[0011] b) polynucleotide that is at least 70% identical to apolynucleotide coding for a polypeptide, that contains the amino acidsequence of SEQ ID No. 3,

[0012] c) polynucleotide coding for a polypeptide, that contains anamino acid sequence that is at least 70% identical to the amino acidsequence of SEQ ID No. 2,

[0013] d) polynucleotide coding for a polypeptide, that contains anamino acid sequence that is at least 70% identical to the amino acidsequence of SEQ ID No. 3,

[0014] e) polynucleotide that is complementary to the polynucleotides ofa), b), c) or d), and

[0015] f) polynucleotide containing at least 15 successive nucleotidesof the polynucleotide sequence of a), b), c), d) or e),

[0016] the polypeptide preferably exhibiting the activity ofsuccinyl-CoA synthetase.

[0017] The present invention also provides the polynucleotide accordingto claim 1, which is preferably a replicable DNA containing:

[0018] (i) the nucleotide sequence shown in SEQ ID No. 1, or

[0019] (ii) at least one sequence that corresponds to the sequence (i)within the region of degeneration of the genetic code, or

[0020] (iii) at least one sequence that hybridizes with the sequencecomplementary to the sequence (i) or (ii), and optionally

[0021] (iv) functionally neutral sense mutations in (i).

[0022] The invention furthermore provides:

[0023] a polynucleotide according to claim 4, containing the nucleotidesequence as shown in SEQ ID No. 1,

[0024] a polynucleotide according to claim 1, wherein the polynucleotideis a preferably recombinant DNA replicable in coryneform bacteria,

[0025] a vector containing parts of the polynucleotide according to theinvention, but at least 15 successive nucleotides of the claimedsequence,

[0026] and coryneform bacteria in which the sucC- and/or sucD-geneis/are attenuated in particular by an insertion or deletion.

[0027] The present invention moreover provides polynucleotides thatsubstantially comprise a polynucleotide sequence, that can be obtainedby screening a corresponding gene library by means of hybridization,that contains the complete sucC- and/or sucD-gene with thepolynucleotide sequence corresponding to SEQ ID No. 1 with a probe thatcontains the sequence of the aforementioned polynucleotide according toSEQ ID No. 1 or a fragment thereof, and isolation of the aforementionedDNA sequence.

[0028] Polynucleotides that contain the sequences according to theinvention are suitable as hybridization probes for RNA, cDNA and DNA, inorder to isolate cDNA, nucleic acids and/or polynucleotides or genes intheir full length that code for succinyl-CoA synthetase, and to isolatesuch cDNA or genes whose sequence has a high similarity to that of thesuccinyl-CoA synthetase genes.

[0029] Polynucleotides that contain the sequences according to theinvention are furthermore suitable as primers, by means of which DNA canbe produced by the polymerase chain reaction (PCR) from genes that codefor succinyl-CoA synthetase.

[0030] Such oligonucleotides serving as probes or primers contain atleast 30, preferably at least 20, and most particularly preferably atleast 15 successive nucleotides. Nucleotides with a length of at least40 or 50 nucleotides are also suitable.

[0031] “Isolated” means separated from its natural environment.

[0032] “Polynucleotide” refers in general to polyribonucleotides andpolydeoxyribonucleotides, in which connection these terms may refer tounmodified RNA or DNA or modified RNA or DNA.

[0033] By the term “polypeptides” are understood peptides or proteinsthat contain two or more amino acids bound via peptide bonds.

[0034] The polypeptides according to the invention include thepolypeptides according to SEQ ID No. 2 and SEQ ID No. 3, in particularthose having the biological activity of succinyl-CoA synthetase as wellas those that are at least 70% identical to the polypeptide according toSEQ ID No. 2 or SEQ ID No. 3, and preferably at least 80% andparticularly preferably at least 90% to 95% identical to the polypeptideaccording to SEQ ID No. 2 or SEQ ID No. 3 and that have theaforementioned activity.

[0035] The present invention furthermore relates to a process for thefermentative production of amino acids selected from the groupcomprising L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine,L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucin,L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan andL-arginine, in particular L-lysine and L-glutamate, using coryneformbacteria that in particular already produce the amino acids, especiallyL-lysine and/or L-glutamate, and in which the nucleotide sequencescoding for the sucC- and/or sucD-gene are attenuated, and in particularare expressed at a low level.

[0036] The term “attenuation” describes in this connection the reductionor switching off of the intracellular activity of one or more enzymes(proteins) in a microorganism that can be coded by the correspondingDNA, by for example using a weak promoter or a gene and/or allele thatcodes for a corresponding enzyme with a low activity and/or inactivatesthe corresponding gene and/or allele or enzyme (protein) and optionallycombines these features.

[0037] The microorganisms that are the subject of the present inventioncan produce amino acids, in particular L-lysine, from glucose, sucrose,lactose, fructose, maltose, molasses, starch, cellulose or from glyceroland ethanol. The microorganisms may be types of coryneform bacteria, inparticular of the genus Corynebacterium. In the genus Corynebacteriumthere should in particular be mentioned the type Corynebacteriumglutamicum, which is known to those skilled in the art for its abilityto produce L-amino acids.

[0038] Suitable strains of the genus Corynebacterium, in particular ofthe type Corynebacterium glutamicum, are in particular the followingknown wild type strains

[0039]Corynebacterium glutamicum ATCC13032

[0040]Corynebacterium acetoglutamicum ATCC15806

[0041]Corynebacterium acetoacidophilum ATCC13870

[0042]Corynebacterium melassecola ATCC17965

[0043]Corynebacterium thermoaminogenes FERM BP-1539

[0044]Brevibacterium flavum ATCC14067

[0045]Brevibacterium lactofermentum ATCC13869 and

[0046]Brevibacterium divaricatum ATCC14020

[0047] and mutants and/or strains obtained therefrom that produceL-amino acids, such as for example the L-lysine-producing strains:

[0048]Corynebacterium glutamicum FERM-P 1709

[0049]Brevibacterium flavum FERM-P 1708

[0050]Brevibacterium lactofermentum FERM-P 1712

[0051]Corynebacterium glutamicum FERM-P 6463

[0052]Corynebacterium glutamicum FERM-P 6464 and

[0053]Corynebacterium glutamicum DSM 5714.

[0054] The new genes sucC and sucD coding for the enzyme succinyl-CoAsynthetase (EC 6.2.1.5) have been isolated from C. glutamicum.

[0055] In order to isolate the sucC- and/or the sucD-gene or also othergenes from C. glutamicum, a gene library of this microorganism is firstof all cultivated in E. coli. The cultivation of gene libraries isdescribed in generally known textbooks and handbooks. By way of examplethere may be mentioned the textbook by Winnacker: Gene und Klone, EineEinführung in die Gentechnologie (Verlag Chemie, Weinheim, Germany,1990) or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A verywell-known gene library is that of the E. coli K-12 strain W3110, whichhas been cultivated by Kohara et al. (Cell 50, 495-508 (1987)) inλ-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265,1996) describe a gene library from C. glutamicum ATCC13032 that has beencultivated with the aid of the cosmid vector SuperCos I (Wahl et al.,1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164)in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic AcidsResearch 16:1563-1575).

[0056] Börmann et al. (Molecular Microbiology 6(3), 317-326 (1992)) inturn describe a gene library obtained from C. glutamicum ATCC13032 usingthe cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). O'Donohue(The Cloning and Molecular Analysis of Four Common Aromatic Amino AcidBiosynthetic Genes from Corynebacterium glutamicum. Ph.D. Thesis,National University of Ireland, Galway, 1997) describes the cloning ofC. glutamicum genes using the λ Zap Expression system described by Shortet al. (Nucleic Acids Research, 16: 7583).

[0057] In order to produce a gene library from C. glutamicum in E. coli,plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) orpUC9 (Vieira et al., 1982, Gene, 19:259-268) may also be used.Particularly suitable as hosts are those E. coli strains that arerestriction-defective and recombinant-defective, such as for example thestrain DH5α (Jeffrey H. Miller: “A Short Course in Bacterial Genetics, ALaboratory Manual and Handbook for Escherichia coli and RelatedBacteria”, Cold Spring Harbour Laboratory Press, 1992).

[0058] The long DNA fragments cloned with the aid of cosmids or otherλ-vectors may then in turn be sub-cloned into accessible vectorssuitable for DNA sequencing.

[0059] Methods for DNA sequencing are described inter alia by Sanger etal. (Proceedings of the National Academy of Sciences of the UnitedStates of America, 74:5463-5467, 1977).

[0060] The DNA sequences that are obtained may then be investigated withknown algorithms and/or sequence analysis programs, such as for examplethat of Staden (Nucleic Acids Research 14, 217-232 (1986)), theGCG-program of Butler (Methods of Biochemical Analysis 39, 74-97(1998)), the FASTA algorithm of Pearson and Lipman (Proceedings of theNational Academy of Sciences USA 85, 2444-2448 (1988)) or the BLASTalgorithm of Altschul et al. (Nature Genetics 6, 119-129 (1994)) andcompared with the sequence entries listed in publicly accessible databanks. Publicly accessible data banks for nucleotide sequences are forexample those of the European Molecular Biologies Laboratories (EMBL,Heidelberg, Germany) or those of the National Center for BiotechnologyInformation (NCBI, Bethesda, Md., USA).

[0061] The new DNA sequences of C. glutamicum coding for the sucC- andsucD-genes have been discovered, and as SEQ ID No. 1 are part of thepresent invention. The amino acid sequence of the corresponding proteinshas furthermore been derived from the existing DNA sequences using themethods described above. The resultant amino acid sequences of the sucC-and sucD-gene product are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0062] Coding DNA sequences that arise from SEQ ID No. 1 due to thedegeneracy of the genetic code are also a subject of the invention. Inthe same way DNA sequences that hybridize with SEQ ID No. 1 or parts ofSEQ ID No. 1 are a subject of the invention. Finally, DNA sequences thatare produced by the polymerase chain reaction (PCR) using primersobtained from SEQ ID No. 1 are also the subject of the invention.

[0063] The person skilled in the art will find information onidentifying DNA sequences by means of hybridization in, inter alia, thehandbook “The DIG System User's Guide for Filter Hybridization”published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and inLiebl et al. (International Journal of Systematic Bacteriology (1991)41: 255-260). The hybridization takes place under stringent conditions,in other words only hybrids are formed in which the probe and targetsequence, i.e. the polynucleotides treated with the probe, are at least70% identical. It is known that the thoroughness of the hybridizationincluding the washing stages is influenced or even determined by varyingthe buffer composition, temperature and the salt concentration. Thehybridization reaction is preferably carried out at a relatively lowdegree of thoroughness compared to the washing stages (HybaidHybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

[0064] A 5×SSC-buffer for example may be used at a temperature of ca.50-68° C. for the hybridization reaction. In this connection probes mayalso be hybridized with polynucleotides that have less than 70% identitywith the sequence of the probe. Such hybrids are less stable and areremoved by washing under stringent conditions. This may be effected forexample by reducing the salt concentration to 2×SSC and optionallysubsequently to 0.5×SSC (The DIG System User's Guide for FilterHybridization, Boehringer Mannheim, Mannheim, Germany, 1995), atemperature of ca. 50-68° C. being maintained. It is also optionallypossible to reduce the salt concentration down to 0.1×SSC. By stepwiseraising of the hybridization temperature in steps of ca. 1-2° C. from 50to 68° C., polynucleotide fragments can be separated that exhibit forexample at least 70% or at least 80% or at least 90% to 95% identity tothe sequence of the probe that is used. Further instructions forhybridization are available on the market in the form of so-called kits(e.g. DIG Easy Hyb von der Firma Roche Diagnostics GmbH, Mannheim,Germany, Catalog No. 1603558).

[0065] The person skilled in the art can find details of the enhancementof DNA sequences by means of the polymerase chain reaction (PCR) in,inter alia, the handbook by Gait: oligonucleotide synthesis: A PracticalApproach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR(Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

[0066] It has now been found that coryneform bacteria produce L-aminoacids, in particular L-lysine, in an improved manner after attenuationof the sucC- and/or sucD-gene.

[0067] In order to achieve such an attenuation, either the expression ofthe sucC- and/or sucD-gene or the catalytic properties of the enzymeproteins can be reduced or switched off. Both measures may optionally becombined.

[0068] The reduction of the gene expression may be achieved by suitableculture conditions or by genetic alteration (mutation) of the signalstructures of the gene expression. Signal structures of the geneexpression are for example repressal genes, activator genes, operators,promoters, attenuators, ribosone bonding sites, the start codon andterminators. The person skilled in the art can find information on theabove in for example patent application WO 96/15246, in Boyd and Murphy(Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss(Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer(Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al.(Microbiology 142: 1297 (1996)) and in known textbooks on genetics andmolecular biology, such as for example the textbook by Knippers(“Molekulare Genetik”, 6^(th) Edition, Georg Thieme Verlag, Stuttgart,Germany, 1995) or the textbook by Winnacker (“Gene und Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990).

[0069] Mutations that lead to an alteration and/or reduction of thecatalytic properties of enzyme proteins are known in the prior art;there may be mentioned by way of example the work carried out by Qiu andGoodman (Journal of Biological Chemistry 272: 8611-8617 (1997)),Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762(1997)) and Möckel (“Die Threonindehydratase aus Corynebacteriumglutamicum: Aufhebung der allosterischen Regulation und Struktur desEnzyms”, reports of the Jülichs Research Centre, Jül-2906, ISSN09442952,Jülich, Germany, 1994). Overviews and summaries may be obtained fromknown textbooks on genetics and molecular biology, such as for examplethose by Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag,Stuttgart, 1986).

[0070] Mutations cover such phenomena as transitions, transversions,insertions and deletions. Depending on the effect of the amino acidexchange on the enzyme activity, one speaks of missense mutations ornonsense mutations. Insertions or deletions of at least one base pair ina gene lead to frame shift mutations, as a result of which false aminoacids are incorporated or the translation is prematurely arrested.Deletions of several codons typically lead to a complete suppression ofthe enzyme activity. Details of producing such mutations are part of theprior art and can be obtained from known textbooks on genetics andmolecular biology, such as for example the textbook by Knippers(“Molekulare Genetik”, 6^(th) Edition, Georg Thieme Verlag, Stuttgart,Germany, 1995), that by Winnacker (“Gene und Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

[0071] A conventional method of mutating genes of C. glutamicum is themethod of gene disruption and gene replacement described by Schwarzerand Pühler (Bio/Technology 9, 84-87 (1991)).

[0072] In the method of gene disruption a central part of the codingregion of the gene that is of interest is cloned in a plasmid vectorthat can replicate in a host (typically E. coli), but not in C.glutamicum. Vectors that may be used include for example pSUP301 (Simonet al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäferet al., Gene 145, 69-73 (1994)), pK18mobsacB or pK19mobsacB (Jäger etal., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (PromegaCorporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal ofBiological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR@Blunt(Firma Invitrogen, Groningen, Niederlande; Bernard et al., Journal ofMolecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991,Journal of Bacteriology 173:4510-4516). The plasmid vector that containsthe central part of the coding region of the gene is then converted byconjugation or transformation into the desired strain of C. glutamicum.

[0073] The method of conjugation is described for example in Schafer etal. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methodsfor transformation are described for example in Thierbach et al.(Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican andShivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a crossover event, the coding region of theaffected gene is disrupted by the vector sequence and two incompletealleles are obtained, each of which lacks the 3′- and the 5′-end. Thismethod has been used for example by Fitzpatrick et al. (AppliedMicrobiology and Biotechnology 42, 575-580 (1994)) in order to switchoff the recA-gene of C. glutamicum. The sucC- and/or sucD-gene may beswitched off in this way.

[0074] In the method of gene replacement a mutation, such as for examplea deletion, insertion or base exchange is produced in vitro in the genethat is of interest. The allele that is produced is in turn cloned in avector that is not replicative for C. glutamicum and the vector is thenconverted by transformation or conjugation into the desired host for C.glutamicum. The incorporation of the mutation and/or of the allele inthe target gene and/or in the target sequence is achieved afterhomologous recombination by means of a first crossover event effectingintegration and an appropriate second crossover event effectingexcision. This method has been used for example by Peters-Wendisch(Microbiology 144, 915-927 (1998)) in order to switch off the pyc-geneof C. glutamicum by means of a deletion. A deletion, insertion or a baseexchange can be incorporated into the sucC- and/or sucD-gene in thisway.

[0075] A deletion, insertion or a base exchange can be incorporated intothe sucC- and/or sucD-gene in this way.

[0076] Furthermore, it was found that by means of one or more amino acidreplacements in the sucC-protein (SEQ ID No. 2) selected from the group:replacement at position 22 by any other proteinogenic amino acid exceptL-proline, replacement at position 44 by any other proteinogenic aminoacid except glycine, and replacement at position 170 by any otherproteinogenic amino acid except L-alanine, an attenuation takes placeand coryneform bacteria that carry the corresponding amino acidreplacement produced amino acids in an improved way, in particularL-lysine and/or L-glutamic acid.

[0077] Particularly preferred are one or more amino acid replacementsselected from the group: L-proline at position 22 by L-serine, glycineat position 44 by L-glutamic acid, and L-alanine at position 170 byL-threonine.

[0078] Most particularly preferred is an SucC-protein that containsL-serine at position 22, L-glutamic acid at position 44, and L-threonineat position 170, as shown in SEQ ID No. 5.

[0079] As shown in SEQ ID No. 4 the replacement of L-proline byL-threonine at position 22 of the amino acid sequence may preferably beeffected by replacing the nucleobase cytosine at position 64 by thymine,the replacement of glycine by L-glutamic acid at position 44 of theamino acid sequence may preferably be effected by replacing thenucleobase guanine at position 131 by adenine, and the replacement ofL-alanine by L-threonine at position 170 of the amino acid sequence maypreferably be effected by replacing the nucleobase guanine at position508 by adenine.

[0080] Conventional mutagenesis methods may be employed for themutagenesis, using mutagenic agents such as for exampleN-methyl-N′-nitro-N-nitrosoguanidine or ultraviolet light.

[0081] Furthermore, in vitro methods may be used for the mutagenesis,such as for example a treatment with hydroxylamine (J. H. Miller: AShort Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1992) or mutagenic oligonucleotides (T. A. Brown:Gentechnologie für Einsteiger, Spektrum Akademischer Verlag, Heidelberg,1993) or the polymerase chain reaction (PCR), as described in thehandbook by Newton and Graham (PCR, Spektrum Akademischer Verlag,Heidelberg, 1994).

[0082] The corresponding sucC alleles and genes are sequenced andincorporated into suitable hosts by for example the method of genereplacement.

[0083] The present invention accordingly also provides coryneformbacteria containing the SucC proteins, in which the amino acid sequenceshown in SEQ ID No. 2 contain one or more replacements selected from thegroup: replacement at position 22 by any other proteinogenic amino acidexcept L-proline, replacement at position 44 by any other proteinogenicamino acid except glycine, and replacement at position 170 by any otherproteinogenic amino acid except L-alanine.

[0084] The invention accordingly furthermore provides polynucleotidesequences derived from coryneform bacteria that contain the genes oralleles coding for the aforementioned SucC proteins.

[0085] Furthermore it may be advantageous for the production of L-aminoacids, in particular L-lysine, in addition to enhance, in particular toover-express, one or more enzymes of the relevant biosynthesis pathway,glycolysis, anaplerosis, citric acid cycle or amino acid export, inorder to attenuate the sucC- and/or sucD-gene.

[0086] The expression “enhancement” describes in this connectionincreasing the intracellular activity of one or more enzymes (proteins)in a microorganism that are coded by the corresponding DNA, by forexample increasing the number of copies of the gene or genes or alleles,using a strong promoter or a gene or allele that codes with a highdegree of activity for a corresponding enzyme (protein), and optionallycombining these measure.

[0087] Thus, in the production of L-lysine and/or L-glutamate, inaddition to the attenuation of the sucC- and/or sucD-gene, one or moreof the genes selected from the following group may be enhanced, inparticular over-expressed:

[0088] the dapA-gene coding for dihydrodipicolinate-synthase (EP-B 0 197335),

[0089] the gap-gene coding for glyceraldehyde-3-phosphate dehydrogenase(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0090] the gene tpi coding for triosephosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0091] the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0092] the pyc-gene coding for pyruvate carboxylase (Eikmanns (1992),Journal of Bacteriology 174:6076-6086),

[0093] the mqo-gene coding for malate:quinone oxidoreductase (Molenaaret al., European Journal of Biochemistry 254, 395-403 (1998)),

[0094] the gene lysC coding for a feed-back resistant aspartate kinase(EP-B-0387527; EP-A-0699759; WO 00/63388)),

[0095] the lysE-gene coding for the L-lysine-export (DE-A-195 48 222),

[0096] the gene zwa1 coding for the Zwa1-protein (DE: 19959328.0, DSM13115).

[0097] Moreover, it may be advantageous for the production of L-lysineand/or L-glutamate, in addition to the attenuation of the sucC- and/orsucD-gene, at the same time to attenuate, in particular to reduce theexpression of one or more of the genes selected from the groupcomprising:

[0098] the gene pck coding for phosphoenolpyruvate-carboxykinase (DE 19950 409.1, DSM 13047),

[0099] the gene pgi coding for glucose-6-phosphate isomerase (U.S. Ser.No. 09/396,478, DSM 12969),

[0100] the gene poxB coding for pyruvate-oxidase (DE:1995 1975.7, DSM13114),

[0101] the gene zwa2 coding for the zwa2-protein (DE: 19959327.2, DSM13113).

[0102] Furthermore it may be advantageous for the production of aminoacid, in particular L-lysine and/or L-glutamate, in addition to theattenuation of the sucC- and/or sucD-gene to switch off undesirablesecondary reactions (Nakayama: “Breeding of Amino Acid ProducingMicroorganisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0103] The microorganisms containing the polynucleotide according toclaim 1 are also the subject of the invention and may be culturedcontinuously or batchwise in a batch process (batch cultivation) or in afed batch or repeated fed batch process in order to produce L-aminoacids, in particular L-lysine. An overview of known cultivation methodsis given in the textbook by Chmiel (Bioprozesstechnik 1. Einführung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[0104] The culture medium to be used must suitably satisfy the demandsof the relevant strains. Descriptions of culture media for variousmicroorganisms are given in the handbook “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981).

[0105] As carbon source there may be used sugars and carbohydrates suchas for example glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats such as for example soya bean oil,sunflower oil, groundnut oil and coconut oil, fatty acids such as forexample palmitic acid, stearic acid and linoleic acid, alcohols such asfor example glycerol and ethanol, and organic acids such as for exampleacetic acid. These substances may be used individually or as a mixture.

[0106] As nitrogen source there may be used organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,corn steep liquor, soya bean flour and urea, or inorganic compounds suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources may be usedindividually or as a mixture.

[0107] As phosphorus source there may be used phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate, or thecorresponding sodium-containing salts. The culture medium mustfurthermore contain salts of metals such as for Example magnesiumsulfate or iron sulfate that are necessary for growth. Finally,essential growth substances such as amino acids and vitamins may, inaddition to the substances mentioned above, be used. Apart from this,suitable precursors may be added to the culture medium. Theaforementioned feedstock substances may be added to the culture in theform of a one-off addition, or may be metered in during the actualcultivation in a suitable way.

[0108] Alkaline compounds such as sodium hydroxide, potassium hydroxide,ammonia or ammonia water or acidic compounds such as phosphoric acid orsulfuric acid may be used in an appropriate manner in order to regulatethe pH of the culture. Antifoaming agents such as for example fatty acidpolyglycol esters may be used to prevent foam formation. Suitableselectively acting substances such as for example antibiotics may beadded to the medium in order to maintain the stability of plasmids.Oxygen or oxygen-containing gas mixtures such as for example air areintroduced into the culture in order to maintain aerobic conditions. Thetemperature of the culture is normally 20° C. to 45° C. and preferably25° C. to 40° C. The culture is continued until a maximum yield of thedesired product has been formed. This target is normally achieved within10 hours to 160 hours.

[0109] A pure culture of the strain Escherichia coliDH5αmcr/pK18mobsacBsucDdel was filed according to the BudapestConvention on 29 Sep. 2000 as DSM 13749 at the German Collection forMicroorganisms and Cell Cultures (DSMZ, Brunswick, Germany).

[0110] A pure culture of the strain Escherichia coliTop10/pCRBluntsucCint was filed according to the Budapest Convention on29 Sep. 2000 as DSM 13750 at the German Collection for Microorganismsand Cell Cultures (DSMZ, Brunswick, Germany).

[0111] Methods for determining L-amino acids are known from the priorart. The analysis may be carried out as described for example bySpackman et al. (Analytical Chemistry, 30, (1958), 1190) by anionexchange chromatography followed by ninhydrin derivatisation or may becarried out by reverse phase HPLC, as described by Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

[0112] The present invention is described in more detail hereinafterwith the aid of embodiments.

EXAMPLE 1

[0113] Production of a genomic cosmid gene library from Corynebacteriumglutamicum ATCC 13032

[0114] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 wasisolated as described by Tauch et al. (1995, Plasmid 33:168-179) andpartially cleaved with the restriction enzyme Sau3AI (AmershamPharmacia, Freiburg, Germany, Product Description Sau3AI, Code no.27-0913-O₂). The DNA fragments were dephosphorylated with shrimpalkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany,Product Description SAP, Code no. 1758250). The DNA of the cosmid vectorSuperCos1 (Wahl et al. (1987) Proceedings of the National Academy ofSciences, USA 84:2160-2164), obtained from Stratagene (La Jolla, USA,Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301) wascleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg,Germany, Product Description XbaI, Code no. 27-0948-O₂) and likewisedephosphorylated with shrimp alkaline phosphatase. The cosmid-DNA wasthen cleaved with the restriction enzyme BamHI (Amersham Pharmacia,Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). Thecosmid-DNA treated in this way was mixed with the treated ATCC13032-DNAand the batch was treated with T4-DNA-ligase (Amersham Pharmacia,Freiburg, Germany, Product Description T4-DNA-ligase, Codeno.27-0870-04). The ligation mixture was then packed in phages with theaid of the Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA,Product Description Gigapack II XL Packing Extract, Code no. 200217). Inorder to infect the E. coli strain NM554 (Raleigh et al. 1988, NucleicAcid Res. 16:1563-1575) the cells were taken up in 10 mM MgSO₄ and mixedwith an aliquot of the phage suspension. Infection and titration of thecosmid bank were carried out as described by Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the cellshaving been plated out on LB-agar (Lennox, 1955, Virology, 1:190) with100 μg/ml ampicillin. Recombinant individual clones were selected afterincubation overnight at 37° C.

EXAMPLE 2

[0115] Isolation and Sequencing of the Genes sucC and sucD

[0116] The cosmid-DNA of an individual colony was isolated using theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)according to the manufacturer's instructions and partially cleaved withthe restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Product No.1758250).

[0117] After gel electrophoresis separation the cosmid fragments wereisolated in the large region from 1500 to 2000 bp using the QiaExII GelExtraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA ofthe sequencing vector pZero-1 obtained from Invitrogen (Groningen,Niederlande, Product Description Zero Background Cloning Kit, ProductNo. K2500-01) was cleaved with the restriction enzyme BamHI (AmershamPharmacia, Freiburg, Germany, Product Description BamHI, Product No.27-0868-04). The ligation of the cosmid fragments in the frequencingvector pZero-1 was carried out as described by Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the DNAmixture having been incubated overnight with T4-ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture was electroporatedinto the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) (Tauch et al. 1994,FEMS Microbiol Letters, 123:343-7) and was plated out on LB-agar(Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin. The plasmidpreparation of the recombinant clones was performed with Biorobot 9600(Product No. 900200, Qiagen, Hilden, Germany). The sequencing wascarried out according to the dideoxy chain termination method of Sangeret al. (1977, Proceedings of the National Academies of Sciences U.S.A.,74:5463-5467) as modified by Zimmermann et al. (1990, Nucleic AcidsResearch, 18:1067). The RR dRhodamin Terminator Cycle Sequencing Kitfrom PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany)was used. The gel electrophoresis separation and analysis of thesequencing reaction was performed in a rotiphoresis NFacrylamide/bisacrylamide gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) together with the “ABI Prism 377” sequencingequipment from PE Applied Biosystems (Weiterstadt, Germany).

[0118] The raw sequence data that were obtained were then processedusing the Staden program package (1986, Nucleic Acids Research,14:217-231) Version 97-0. The individual sequences of the pZerolderivates were assembled into a coherent Contig. The computer-assistedanalysis of the coding region was performed with the program XNIP(Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyseswere carried out with the BLAST search programs (Altschul et al., 1997,Nucleic Acids Research, 25:3389-3402), against the non-redundant databank of the National Center for Biotechnology Information (NCBI,Bethesda, Md., USA).

[0119] The nucleotide sequence that was obtained is illustrated in SEQID No. 1. Analysis of the nucleotide sequence showed an open readingframe of 1206 base pairs, which was identified as sucC-gene, as well asan open reading frame of 882 base pairs, identified as sucD. ThesucC-gene codes for a polypeptide of 402 amino acids, which is shown inSEQ ID No. 2. The sucD-gene codes for a polypeptide of 294 amino acids,which is shown in SEQ ID No. 3.

EXAMPLE 3

[0120] 3.1 Production of an Integration Vector for the IntegrationMutagenesis of the sucC-Gene

[0121] Chromosomal DNA was isolated from the strain ATCC 13032 accordingto the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)).On the basis of the sequence of the sucC-gene for C. glutamicum knownfrom Example 1 the following oligonucleotides were selected for thepolymerase chain reaction (see SEQ ID No. 6 and SEQ ID No. 7): primersucC-in1: 5′CGC GCG AAT CGT TCG TAT 3′ primer sucC-in2: 5′CGC CAC CAATGT CTA GGA 3′

[0122] The indicated primers were synthesised by MWG Biotech (Ebersberg,Germany) and the PCR reaction was carried out with the Pwo polymerasefrom Boehringer Mannheim (Germany, Product Description Pwo DNAPolymerase, Product No. 1 644 947) according to the standard PCR methodof Innis et al. (PCR Protocols. A Guide to Methods and Applications,1990, Academic Press). With the aid of the polymerase chain reaction theprimers permit the enhancement of an approximately 0.55 kb largeinternal fragment of the sucC-gene. The product enhanced in this way waschecked by electrophoresis in a 0.8% agarose gel.

[0123] The enhanced DNA fragment was ligated into the vector pCR®BluntII (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993))using the Zero Blunt™ Kit from Invitrogen Corporation (Carlsbad, Calif.,USA; Catalogue Number K2700-20).

[0124] The E. coli strain TOP10 was then electroporated into theligation batch (Hanahan, In: DNA Cloning. A Practical Approach, Vol. I,IRL-Press, Oxford, Washington D.C., USA, 1985). The selection ofplasmid-carrying cells was performed by plating out the transformationbatch onto LB agar (Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) that had been supplemented with 25 mg/l ofkanamycin. Plasmid DNA was isolated from a transformant with the aid ofthe QIAprep Spin Miniprep Kit from Qiagen and checked by restrictionwith the restriction enzyme EcoRI followed by agarose gelelectrophoresis (0.8%). The plasmid was named pCRBluntsucCint and isshown in FIG. 1.

[0125] 3.2 Deletion of the sucD-Gene

[0126] For this purpose chromosomal DNA was isolated from the strainATCC13032 by the method of Tauch et al. (1995, Plasmid 33:168-179). Onthe basis of the sequence of the sucD-gene for C. glutamicum known fromExample 2 the oligonucleotides described hereinafter were selected forproducing the sucD deletion allele (see SEQ ID No. 8 to SEQ ID No. 11):primer sucD-d1: 5′-CGA TGT GAT TGC GCT TGA TG -3′ deletion primersucD-d2: 5′-ACC TCA CGC ATA AGC TTC GCA TGC TCT GAA CCT TCC GAA C -3′deletion primer sucD-d3: 5′-GTT CGG AAG GTT CAG AGC ATG CGA AGC TTA TGCGTG AGG T -3′ primer sucD-d4: 5′-ATG AAG CCA GCG ACT GCA GA -3′

[0127] The relevant primers were synthesised by MWG Biotech (Ebersberg,Germany) and the PCR reaction was carried out using the Pfu polymerase(Stratagene, Product. No. 600135, La Jolla, USA) and the PTC100-Thermocyclers (MJ Research Inc., Waltham, USA). With the aid of thepolymerase chain reaction the primers permit the enhancement of a sucDallele with internal deletion. The product enhanced in this way wastested by electrophoresis in a 0.8% agarose gel and was also sequencedas described by Sanger et al. (Proceedings of the National Academy ofSciences of the United States of America, 74:5463-5467, 1977).

EXAMPLE 4

[0128] 4.1 Integration Mutagenesis of the sucC-Gene in the Strain DSM5715

[0129] The vector pCRBluntsucCint mentioned in Example 3.1 waselectroporated into C. glutamicum DSM 5715 (EP 0 435 132) according tothe electroporation method of Tauch et. al. (FEMS MicrobiologicalLetters, 123:343-347 (1994)). The strain DSM 5715 is an AEC resistantL-lysine producer. The vector pCRBlunt-sucCint cannot independentlyreplicate in DSM5715 and accordingly only remains in the cellulose if ithad integrated into the chromosome of DSM 5715. The selection of cloneswith pCRBluntsucCint integrated into the chromosome is performed byplating out the electroporation batch onto LB agar (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) that had been supplementedwith 15 mg/l of kanamycin.

[0130] In order to detect the integration the sucCint fragment waslabelled according to the method described in “The DIG System User'sGuide for Filter Hybridization” of Boehringer Mannheim GmbH (Mannheim,Germany, 1993) using the Dig-Hybridization Kit from Boehringer.Chromosomal DNA of a potential integrant was isolated according to themethod of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and wascut in each case with the restriction enzyme SphI and HindIII. Theresultant fragments were separated by means of agarose gelelectrophoresis and hybridized at 68° C. using the Dig-Hybridization Kitfrom Boehringer. The plasmid pCRBluntsucCint named in Example 3.1 hadinserted itself into the chromosome of DSM5715 within the chromosomalsucC-gene. The strain was identified as DSM5715::pCRBluntsucCint.

[0131] 4.2 Construction of the Exchange Vector pK18mobsacBsucDdel

[0132] The sucD-deletion derivative obtained in Example 3.2 was, afterseparation in an agarose gel (0.8%) using the Qiagenquick Gel ExtractionKit (Qiagen, Hilden, Germany), isolated from the agarose gel and thenused with the mobilisable cloning vector pK18mobsacB (Schäfer et al.(1994), Gene 14: 69-73) for the ligation. This had previously beencleaved with the restriction enzymes XmaI- and XbaI, mixed with thesucD-deletion allele, and treated with T4-DNA-ligase (AmershamPharmacia, Freiburg, Germany).

[0133] The E. coli strain DH5αmcr (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) was thenelectroporated with the ligation batch (Hanahan, In. DNA Cloning. APractical Approach, Vol.1, ILR-Press, Cold Spring Harbor, N.Y., 1989).The plasmid-carrying cells were selected by plating out thetransformation batch onto LB agar (Sambrock et al., Molecular Cloning: ALaboratory Manual. 2^(nd) Ed. Cold Spring Harbor, N.Y., 1989) that hadbeen supplemented with 25 mg/l of kanamycin.

[0134] Plasmid DNA was isolated from a transformant by means of theQIAprep Spin Miniprep Kit from Qiagen, and the cloned sucD-deletionallele was verified by means of sequencing by the company MWG Biotech(Ebersberg, Germany). The plasmid was named pK18mobsacBsucDdel. Thestrain was identified as E. coliH5αmcr/pK18mobsacBsucDdel.

[0135] 4.3 Deletion Mutagenesis of the sucD-Gene in the C. glutamicumStrain DSM 5715

[0136] The vector pK18mobsacBsucDdel mentioned in Example 4.2 waselectroporated according to the electroporation method of Tauch et al.,(1989 FEMS Microbiology Letters 123: 343-347). The vector cannotreplicate independently in DSM 5715 and accordingly only remains in thecellulose if it has integrated into the chromosome. The selection ofclones with integrated pK18mobsacBsucDdel was performed by plating outthe electroporation batch onto LB-agar (Sambrock et al., MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor, N.Y.,1989) that had been supplemented with 15 mg/l of kanamycin. Cultivatedclones were streaked out onto LB-agar plates containing 25 mg/l ofkanamycin and incubated for 16 hours at 33° C.

[0137] In order to achieve the excision of the plasmid together with thecomplete chromosomal copy of the sucD-gene, the clones were then grownon LB-agar containing 10% sucrose. The plasmid pK18mobsacB contains acopy of the sacB-gene, which converts sucrose into levansucrase that isnot toxic for C. glutamicum. Accordingly only those clones in which theintegrated pK18mobsacBsucDdel has in turn been excised can be grown onLB-agar containing sucrose. In the excision either the completechromosomal copy of the sucD-gene or the incomplete copy together withthe internal deletion can be excised together with the plasmid.

[0138] In order to detect whether the incomplete copy of sucD stillremains in the chromosome, the plasmid pK18mobsacBsucDdel fragment waslabelled according to the method described in “The DIG System User'sGuide for Filter Hybridization” published by Boehringer Mannheim GmbH(Mannheim, Germany, 1993) using the Dig-Hybridization Kit fromBoehringer. Chromosomal DNA of a potential deletion mutant was isolatedaccording to the method of Eikmanns et al. (Microbiology 140: 1817-1828(1994)) and was in each case cut into separate sections using therestriction enzymes SphI and PstI. The resultant fragments wereseparated by agarose gel electrophoresis and hybridized at 68° C. usingthe Dig Hybridization Kit from Boehringer. On the basis of the resultantfragments it could be shown that the strain DSM5715 has lost itscomplete copy of the sucD-gene and instead only the deleted copy isstill available.

[0139] The strain was identified as C. glutamicum DSM5715ΔsucD.

EXAMPLE 5

[0140] 5.1 Production of L-Glutamate Using the Strain DSM5715::pCRBluntsucCint

[0141] The C. glutamicum strain DSM5715::pCRBluntsucCint obtained inExample 4.1 was cultivated in a suitable nutrient medium for producingL-glutamate and the glutamate content in the culture supernatant wasdetermined.

[0142] For this purpose the strain was first of all incubated for 24hours at 33° C. on agar plates with the corresponding antibiotic(brain-heart agar with kanamycin (25 mg/l). A pre-culture was inoculatedusing this agar plate culture (10 ml of medium in a 100 ml Erlenmeyerflask). The full medium Cg III was used as medium for the pre-culture.Medium Cg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast Extract 10g/l Glucose (separately autoclaved) 2% (w/v) The pH was adjusted to pH7.4

[0143] Kanamycin (25 mg/l) was added to this medium. The pre-culture wasincubated on a shaker for 16 hours at 33° C. at 240 rpm. A main culturewas inoculated from this pre-culture so that the initial optical density(660 nm) of the main culture was 0.1 OD. The medium MM was used for themain culture. Medium MM CSL (Corn Steep Liquor) 5 g/l MOPS(Morpholinopropanesulfonic 20 g/l acid) Glucose (separately autoclaved)50 g/l Salts: (NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄.7H₂0 1.0 g/lCaCl₂.2H₂0 10 mg/l FeSO₄.7H₂0 10 mg/l MnSO₄.H₂O 5.0 mg/l Biotin (sterilefiltered) 0.3 mg/l Thiamine.HCl (sterile filtered) 0.2 mg/l Fumarate(sterile filtered) 5.81 g/l Leucine (sterile filtered) 0.1 g/l CaCO₃ 25g/l

[0144] CSL, MOPS and the salt solution are adjusted with ammonia waterto pH 7 and autoclaved. The sterile substrate and vitamin solutions aswell as the dry autoclaved CaCO₃ are then added.

[0145] Cultivation takes place in a 10 ml volume in a 100 ml Erlenmeyerflask with baffles. Kanamycin (25 mg/l) was added. Cultivation tookplace at 33° C. and 80% atmospheric humidity.

[0146] After 24 hours the OD was measured at a measurement wavelength of660 nm using the Biomek 1000 instrument (Beckmann Instruments GmbH,Munich). The amount of glutamate formed was measured in an amino acidanalyser from Eppendorf-BioTronik (Hamburg, Germany) by ion exchangechromatography and post-column derivatisation with ninhydrin detection.

[0147] The result of the test is shown in Table 1. TABLE 1 ODL-glutamate Strain (660 nm) mg/l DSM5715 10.4 20 DSM5715::pCRBlunt 3.9154 sucCint

[0148] 5.2 Production of L-Glutamate Using the Strain DSM5715ΔsucD

[0149] The C. glutamicum strain DSM5715/pK18mobsacBsucDdel obtained inExample 4.3 was cultivated in a nutrient medium suitable for producingL-glutamate and the glutamate content in the culture supernatant wasmeasured.

[0150] For this purpose the strain was first of all incubated for 24hours at 33° C. on agar plates. A preculture was inoculated using thisagar plate culture (10 ml medium in 100 ml Erlenmeyer flask). The fullmedium CgIII was used for the preculture. Kanamycin (25 mg/l) was addedto this medium. The preculture was incubated on a shaker for 16 hours at33° C. and at 240 rpm. A main culture was inoculated from thispreculture so that the initial OD (660 nm) of the main culture was 0.1OD. The medium MM was used for the main culture.

[0151] The cultivation was carried out in a 10 ml volume in a 100 mlErlenmeyer flask equipped with baffles. Cultivation was carried out at33° C. and 80% atmospheric humidity.

[0152] After 72 hours the OD was measured at a measurement wavelength of660 nm using a Biomek 1000 instrument (Beckmann Instruments GmbH,Munich). The amount of glutamate formed was measured with an amino acidanalyser from Eppendorf-BioTronik (Hamburg, Germany) by ion exchangechromatography and post-column derivatisation with ninhydrin detection.

[0153] The result of the test is shown in Table 2. TABLE 2 ODL-glutamate Strain (660 nm) mg/l DSM5715 8.1 7 DSM5715ΔsucD 13.3 33

BRIEF DESCRIPTION OF THE FIGURES

[0154]FIG. 1: Map of the plasmid pCRBluntsucCint.

[0155]FIG. 2: Map of the plasmid pK18mobsacBsucDdel

[0156] The acronyms and abbreviations used in FIG. 1 have the followingmeanings: KmR: Kanamycin resistance gene Zeocin: Zeocin resistance geneHindIII Cutting site of the restriction enzyme HindIII SphI Cutting siteof the restriction enzyme SphI EcoRI: Cutting site of the restrictionenzyme EcoRI sucCint: Internal fragment of the sucC-gene ColE1 ori:Replication origin of the plamid ColE1

[0157] The acronyms and abbreviations used in FIG. 2 have the followingmeanings: oriV: ColE1-like origin of pMB1 sacB The sac-B gene coding forthe protein levansucrose RP4mob: RP4-mobilisation site Kan: Resistancegene for kanamycin sucDdel′: 5′-terminal fragment of the sucD-gene fromC. glutamicum sucDdel″: 3′-terminal fragment of the sucD-gene from C.glutamicum SphI: Cutting site of the restriction enzyme SphI PstI:Cutting site of the restriction enzyme PstI XmaI: Cutting site of therestriction enzyme XmaI XbaI: Cutting site of the restriction enzymeXbaI

[0158]

1 5 1 2410 DNA Corynebacterium glutamicum CDS (142)..(1347) sucCsequence 1 gcaccacgga tccaattttg ttgcaatttg caaagtttac agtgttagacttcacaatac 60 gatcatattg gtgagttgaa acacttactt ttacgggaag actttgttaaagacgcagaa 120 ggctctaagc atgggccgga a atg gaa ttg gca gtg gat ctt tttgaa tac 171 Met Glu Leu Ala Val Asp Leu Phe Glu Tyr 1 5 10 caa gca cgggac ctc ttt gaa acc cat ggt gtg cca gtg ttg aag gga 219 Gln Ala Arg AspLeu Phe Glu Thr His Gly Val Pro Val Leu Lys Gly 15 20 25 att gtg gca tcaaca cca gag gcg gcg agg aaa gcg gct gag gaa atc 267 Ile Val Ala Ser ThrPro Glu Ala Ala Arg Lys Ala Ala Glu Glu Ile 30 35 40 ggc gga ctg acc gtcgtc aag gct cag gtc aag gtg ggc gga cgt ggc 315 Gly Gly Leu Thr Val ValLys Ala Gln Val Lys Val Gly Gly Arg Gly 45 50 55 aag gcg ggt ggc gtc cgtgtg gca ccg acg tcg gct cag gct ttt gat 363 Lys Ala Gly Gly Val Arg ValAla Pro Thr Ser Ala Gln Ala Phe Asp 60 65 70 gct gcg gat gcg att ctc ggcatg gat atc aaa gga cac act gtt aat 411 Ala Ala Asp Ala Ile Leu Gly MetAsp Ile Lys Gly His Thr Val Asn 75 80 85 90 cag gtg atg gtg gcg cag ggcgct gac att gct gag gaa tac tat ttc 459 Gln Val Met Val Ala Gln Gly AlaAsp Ile Ala Glu Glu Tyr Tyr Phe 95 100 105 tcc att ttg ttg gat cgc gcgaat cgt tcg tat ctg gct atg tgc tct 507 Ser Ile Leu Leu Asp Arg Ala AsnArg Ser Tyr Leu Ala Met Cys Ser 110 115 120 gtt gaa ggt ggc atg gag atcgag atc ctg gcg aag gaa aag cct gaa 555 Val Glu Gly Gly Met Glu Ile GluIle Leu Ala Lys Glu Lys Pro Glu 125 130 135 gct ttg gca aag gtg gaa gtggat ccc ctc act ggt att gat gag gac 603 Ala Leu Ala Lys Val Glu Val AspPro Leu Thr Gly Ile Asp Glu Asp 140 145 150 aaa gcg cgg gag att gtc actgct gct ggc ttt gaa act gag gtg gca 651 Lys Ala Arg Glu Ile Val Thr AlaAla Gly Phe Glu Thr Glu Val Ala 155 160 165 170 gag aaa gtc att ccg gtgctg atc aag atc tgg cag gtg tat tac gaa 699 Glu Lys Val Ile Pro Val LeuIle Lys Ile Trp Gln Val Tyr Tyr Glu 175 180 185 gag gaa gca aca ctc gttgag gtg aac ccg ttg gtg ctc acg gat gac 747 Glu Glu Ala Thr Leu Val GluVal Asn Pro Leu Val Leu Thr Asp Asp 190 195 200 ggc gat gtg att gcg cttgat ggc aag atc acg ctg gat gat aac gct 795 Gly Asp Val Ile Ala Leu AspGly Lys Ile Thr Leu Asp Asp Asn Ala 205 210 215 gat ttc cgc cat gat aaccgt ggt gcg ttg gct gaa tct gcc ggt ggc 843 Asp Phe Arg His Asp Asn ArgGly Ala Leu Ala Glu Ser Ala Gly Gly 220 225 230 ttg gac att ttg gaa ctgaag gcc aag aag aat gat ctg aac tac gtg 891 Leu Asp Ile Leu Glu Leu LysAla Lys Lys Asn Asp Leu Asn Tyr Val 235 240 245 250 aaa ctt gat ggc tctgtg ggc atc att ggc aat ggt gca ggt ttg gtg 939 Lys Leu Asp Gly Ser ValGly Ile Ile Gly Asn Gly Ala Gly Leu Val 255 260 265 atg tcc acg ttg gatatc gtg gct gca gct ggt gaa cgc cat ggt ggg 987 Met Ser Thr Leu Asp IleVal Ala Ala Ala Gly Glu Arg His Gly Gly 270 275 280 cag cgc ccc gcg aacttc cta gac att ggt ggc gga gca tca gct gaa 1035 Gln Arg Pro Ala Asn PheLeu Asp Ile Gly Gly Gly Ala Ser Ala Glu 285 290 295 tcg atg gct gct ggtctc gat gtg atc ctt ggg gat agc cag gta cgc 1083 Ser Met Ala Ala Gly LeuAsp Val Ile Leu Gly Asp Ser Gln Val Arg 300 305 310 agt gtg ttt gtg aatgtg ttt ggt ggc atc acc gcg tgt gat gtg gtg 1131 Ser Val Phe Val Asn ValPhe Gly Gly Ile Thr Ala Cys Asp Val Val 315 320 325 330 gca aag gga atcgtt gga gct ttg gat gtg ctc ggc gat caa gca acg 1179 Ala Lys Gly Ile ValGly Ala Leu Asp Val Leu Gly Asp Gln Ala Thr 335 340 345 aag cct ctt gtggtg cgc ctt gat ggc aac aac gtg gtg gaa ggc aga 1227 Lys Pro Leu Val ValArg Leu Asp Gly Asn Asn Val Val Glu Gly Arg 350 355 360 cga atc ctc gcggaa tat aac cac cct ttg gtc acc gtt gtg gag ggt 1275 Arg Ile Leu Ala GluTyr Asn His Pro Leu Val Thr Val Val Glu Gly 365 370 375 atg gat gca gcggct gat cac gct gcc cat ttg gcc aat ctt gcc cag 1323 Met Asp Ala Ala AlaAsp His Ala Ala His Leu Ala Asn Leu Ala Gln 380 385 390 cac ggc cag ttcgca acc gct aat tagttaagga gcacctgttt aatc atg 1374 His Gly Gln Phe AlaThr Ala Asn Met 395 400 tct att ttt ctc aat tca gat tcc cgc atc atc attcag ggc att acc 1422 Ser Ile Phe Leu Asn Ser Asp Ser Arg Ile Ile Ile GlnGly Ile Thr 405 410 415 ggt tcg gaa ggt tca gag cat gcg cgt cga att ttagcc tct ggt gcg 1470 Gly Ser Glu Gly Ser Glu His Ala Arg Arg Ile Leu AlaSer Gly Ala 420 425 430 435 aag ctc gtg ggt ggc acc aac ccc cgc aaa gctggg caa acc att ttg 1518 Lys Leu Val Gly Gly Thr Asn Pro Arg Lys Ala GlyGln Thr Ile Leu 440 445 450 atc aat gac act gag ttg cct gta ttt ggc actgtt aag gaa gca atg 1566 Ile Asn Asp Thr Glu Leu Pro Val Phe Gly Thr ValLys Glu Ala Met 455 460 465 gag gaa acg ggt gcg gat gtc acc gta att ttcgtt cct cca gcc ttt 1614 Glu Glu Thr Gly Ala Asp Val Thr Val Ile Phe ValPro Pro Ala Phe 470 475 480 gcc aaa gct gcg atc att gaa gct atc gac gctcac atc cca ctg tgc 1662 Ala Lys Ala Ala Ile Ile Glu Ala Ile Asp Ala HisIle Pro Leu Cys 485 490 495 gtg att att act gag ggc atc cca gtg cgt gacgct tct gag gcg tgg 1710 Val Ile Ile Thr Glu Gly Ile Pro Val Arg Asp AlaSer Glu Ala Trp 500 505 510 515 gct tat gcc aag aag gtg gga cac acc cgcatc att ggc cct aac tgc 1758 Ala Tyr Ala Lys Lys Val Gly His Thr Arg IleIle Gly Pro Asn Cys 520 525 530 cca ggc att att act ccc ggc gaa tct cttgcg gga att acg ccg gca 1806 Pro Gly Ile Ile Thr Pro Gly Glu Ser Leu AlaGly Ile Thr Pro Ala 535 540 545 aac att gca ggt tcc ggc ccg atc ggg ttgatc tca aag tcg gga aca 1854 Asn Ile Ala Gly Ser Gly Pro Ile Gly Leu IleSer Lys Ser Gly Thr 550 555 560 ctg act tat cag atg atg tac gaa ctt tcagat att ggc att tct acg 1902 Leu Thr Tyr Gln Met Met Tyr Glu Leu Ser AspIle Gly Ile Ser Thr 565 570 575 gcg att ggt att ggc ggt gac cca atc atcggt aca acc cat atc gac 1950 Ala Ile Gly Ile Gly Gly Asp Pro Ile Ile GlyThr Thr His Ile Asp 580 585 590 595 gct ctg gag gcc ttt gaa gct gat cctgag acc aag gca atc gtc atg 1998 Ala Leu Glu Ala Phe Glu Ala Asp Pro GluThr Lys Ala Ile Val Met 600 605 610 atc ggt gag atc ggt gga gat gca gaggaa cgc gct gct gac ttc att 2046 Ile Gly Glu Ile Gly Gly Asp Ala Glu GluArg Ala Ala Asp Phe Ile 615 620 625 tct aag cac gtg aca aaa cca gtt gtgggt tac gtg gca ggc ttt acc 2094 Ser Lys His Val Thr Lys Pro Val Val GlyTyr Val Ala Gly Phe Thr 630 635 640 gcc cct gaa gga aag acc atg ggg catgct ggc gcc atc gtg aca ggt 2142 Ala Pro Glu Gly Lys Thr Met Gly His AlaGly Ala Ile Val Thr Gly 645 650 655 tca gaa ggc act gcg cga gca aag aagcat gca ttg gag gcc gtg ggt 2190 Ser Glu Gly Thr Ala Arg Ala Lys Lys HisAla Leu Glu Ala Val Gly 660 665 670 675 gtt cgc gtg gga aca act ccg agtgaa acc gcg aag ctt atg cgt gag 2238 Val Arg Val Gly Thr Thr Pro Ser GluThr Ala Lys Leu Met Arg Glu 680 685 690 gta gtt gca gct ttg taactaacaggccacagatc ttagctttga ccagcggatt 2293 Val Val Ala Ala Leu 695 tgtggctaatcgcccggtct gtgtagagta ttcatctgtg cgcaggacag tgtgacaaac 2353 actgaatagtgcatggcttt aaggccctgt ggcgcagttg gttagcgcgc cgccctg 2410 2 402 PRTCorynebacterium glutamicum 2 Met Glu Leu Ala Val Asp Leu Phe Glu Tyr GlnAla Arg Asp Leu Phe 1 5 10 15 Glu Thr His Gly Val Pro Val Leu Lys GlyIle Val Ala Ser Thr Pro 20 25 30 Glu Ala Ala Arg Lys Ala Ala Glu Glu IleGly Gly Leu Thr Val Val 35 40 45 Lys Ala Gln Val Lys Val Gly Gly Arg GlyLys Ala Gly Gly Val Arg 50 55 60 Val Ala Pro Thr Ser Ala Gln Ala Phe AspAla Ala Asp Ala Ile Leu 65 70 75 80 Gly Met Asp Ile Lys Gly His Thr ValAsn Gln Val Met Val Ala Gln 85 90 95 Gly Ala Asp Ile Ala Glu Glu Tyr TyrPhe Ser Ile Leu Leu Asp Arg 100 105 110 Ala Asn Arg Ser Tyr Leu Ala MetCys Ser Val Glu Gly Gly Met Glu 115 120 125 Ile Glu Ile Leu Ala Lys GluLys Pro Glu Ala Leu Ala Lys Val Glu 130 135 140 Val Asp Pro Leu Thr GlyIle Asp Glu Asp Lys Ala Arg Glu Ile Val 145 150 155 160 Thr Ala Ala GlyPhe Glu Thr Glu Val Ala Glu Lys Val Ile Pro Val 165 170 175 Leu Ile LysIle Trp Gln Val Tyr Tyr Glu Glu Glu Ala Thr Leu Val 180 185 190 Glu ValAsn Pro Leu Val Leu Thr Asp Asp Gly Asp Val Ile Ala Leu 195 200 205 AspGly Lys Ile Thr Leu Asp Asp Asn Ala Asp Phe Arg His Asp Asn 210 215 220Arg Gly Ala Leu Ala Glu Ser Ala Gly Gly Leu Asp Ile Leu Glu Leu 225 230235 240 Lys Ala Lys Lys Asn Asp Leu Asn Tyr Val Lys Leu Asp Gly Ser Val245 250 255 Gly Ile Ile Gly Asn Gly Ala Gly Leu Val Met Ser Thr Leu AspIle 260 265 270 Val Ala Ala Ala Gly Glu Arg His Gly Gly Gln Arg Pro AlaAsn Phe 275 280 285 Leu Asp Ile Gly Gly Gly Ala Ser Ala Glu Ser Met AlaAla Gly Leu 290 295 300 Asp Val Ile Leu Gly Asp Ser Gln Val Arg Ser ValPhe Val Asn Val 305 310 315 320 Phe Gly Gly Ile Thr Ala Cys Asp Val ValAla Lys Gly Ile Val Gly 325 330 335 Ala Leu Asp Val Leu Gly Asp Gln AlaThr Lys Pro Leu Val Val Arg 340 345 350 Leu Asp Gly Asn Asn Val Val GluGly Arg Arg Ile Leu Ala Glu Tyr 355 360 365 Asn His Pro Leu Val Thr ValVal Glu Gly Met Asp Ala Ala Ala Asp 370 375 380 His Ala Ala His Leu AlaAsn Leu Ala Gln His Gly Gln Phe Ala Thr 385 390 395 400 Ala Asn 3 294PRT Corynebacterium glutamicum 3 Met Ser Ile Phe Leu Asn Ser Asp Ser ArgIle Ile Ile Gln Gly Ile 1 5 10 15 Thr Gly Ser Glu Gly Ser Glu His AlaArg Arg Ile Leu Ala Ser Gly 20 25 30 Ala Lys Leu Val Gly Gly Thr Asn ProArg Lys Ala Gly Gln Thr Ile 35 40 45 Leu Ile Asn Asp Thr Glu Leu Pro ValPhe Gly Thr Val Lys Glu Ala 50 55 60 Met Glu Glu Thr Gly Ala Asp Val ThrVal Ile Phe Val Pro Pro Ala 65 70 75 80 Phe Ala Lys Ala Ala Ile Ile GluAla Ile Asp Ala His Ile Pro Leu 85 90 95 Cys Val Ile Ile Thr Glu Gly IlePro Val Arg Asp Ala Ser Glu Ala 100 105 110 Trp Ala Tyr Ala Lys Lys ValGly His Thr Arg Ile Ile Gly Pro Asn 115 120 125 Cys Pro Gly Ile Ile ThrPro Gly Glu Ser Leu Ala Gly Ile Thr Pro 130 135 140 Ala Asn Ile Ala GlySer Gly Pro Ile Gly Leu Ile Ser Lys Ser Gly 145 150 155 160 Thr Leu ThrTyr Gln Met Met Tyr Glu Leu Ser Asp Ile Gly Ile Ser 165 170 175 Thr AlaIle Gly Ile Gly Gly Asp Pro Ile Ile Gly Thr Thr His Ile 180 185 190 AspAla Leu Glu Ala Phe Glu Ala Asp Pro Glu Thr Lys Ala Ile Val 195 200 205Met Ile Gly Glu Ile Gly Gly Asp Ala Glu Glu Arg Ala Ala Asp Phe 210 215220 Ile Ser Lys His Val Thr Lys Pro Val Val Gly Tyr Val Ala Gly Phe 225230 235 240 Thr Ala Pro Glu Gly Lys Thr Met Gly His Ala Gly Ala Ile ValThr 245 250 255 Gly Ser Glu Gly Thr Ala Arg Ala Lys Lys His Ala Leu GluAla Val 260 265 270 Gly Val Arg Val Gly Thr Thr Pro Ser Glu Thr Ala LysLeu Met Arg 275 280 285 Glu Val Val Ala Ala Leu 290 4 1206 DNACorynebacterium glutamicum CDS (1)..(1206) sucC coding sequence 4 atggaa ttg gca gtg gat ctt ttt gaa tac caa gca cgg gac ctc ttt 48 Met GluLeu Ala Val Asp Leu Phe Glu Tyr Gln Ala Arg Asp Leu Phe 1 5 10 15 gaaacc cat ggt gtg tca gtg ttg aag gga att gtg gca tca aca cca 96 Glu ThrHis Gly Val Ser Val Leu Lys Gly Ile Val Ala Ser Thr Pro 20 25 30 gag gcggcg agg aaa gcg gct gag gaa atc ggc gaa ctg acc gtc gtc 144 Glu Ala AlaArg Lys Ala Ala Glu Glu Ile Gly Glu Leu Thr Val Val 35 40 45 aag gct caggtc aag gtg ggc gga cgt ggc aag gcg ggt ggc gtc cgt 192 Lys Ala Gln ValLys Val Gly Gly Arg Gly Lys Ala Gly Gly Val Arg 50 55 60 gtg gca ccg acgtcg gct cag gct ttt gat gct gcg gat gcg att ctc 240 Val Ala Pro Thr SerAla Gln Ala Phe Asp Ala Ala Asp Ala Ile Leu 65 70 75 80 ggc atg gat atcaaa gga cac act gtt aat cag gtg atg gtg gcg cag 288 Gly Met Asp Ile LysGly His Thr Val Asn Gln Val Met Val Ala Gln 85 90 95 ggc gct gac att gctgag gaa tac tat ttc tcc att ttg ttg gat cgc 336 Gly Ala Asp Ile Ala GluGlu Tyr Tyr Phe Ser Ile Leu Leu Asp Arg 100 105 110 gcg aat cgt tcg tatctg gct atg tgc tct gtt gaa ggt ggc atg gag 384 Ala Asn Arg Ser Tyr LeuAla Met Cys Ser Val Glu Gly Gly Met Glu 115 120 125 atc gag atc ctg gcgaag gaa aag cct gaa gct ttg gca aag gtg gaa 432 Ile Glu Ile Leu Ala LysGlu Lys Pro Glu Ala Leu Ala Lys Val Glu 130 135 140 gtg gat ccc ctc actggt att gat gag gac aaa gcg cgg gag att gtc 480 Val Asp Pro Leu Thr GlyIle Asp Glu Asp Lys Ala Arg Glu Ile Val 145 150 155 160 act gct gct ggcttt gaa act gag gtg aca gag aaa gtc att ccg gtg 528 Thr Ala Ala Gly PheGlu Thr Glu Val Thr Glu Lys Val Ile Pro Val 165 170 175 ctg atc aag atctgg cag gtg tat tac gaa gag gaa gca aca ctc gtt 576 Leu Ile Lys Ile TrpGln Val Tyr Tyr Glu Glu Glu Ala Thr Leu Val 180 185 190 gag gtg aac ccgttg gtg ctc acg gat gac ggc gat gtg att gcg ctt 624 Glu Val Asn Pro LeuVal Leu Thr Asp Asp Gly Asp Val Ile Ala Leu 195 200 205 gat ggc aag atcacg ctg gat gat aac gct gat ttc cgc cat gat aac 672 Asp Gly Lys Ile ThrLeu Asp Asp Asn Ala Asp Phe Arg His Asp Asn 210 215 220 cgt ggt gcg ttggct gaa tct gcc ggt ggc ttg gac att ttg gaa ctg 720 Arg Gly Ala Leu AlaGlu Ser Ala Gly Gly Leu Asp Ile Leu Glu Leu 225 230 235 240 aag gcc aagaag aat gat ctg aac tac gtg aaa ctt gat ggc tct gtg 768 Lys Ala Lys LysAsn Asp Leu Asn Tyr Val Lys Leu Asp Gly Ser Val 245 250 255 ggc atc attggc aat ggt gca ggt ttg gtg atg tcc acg ttg gat atc 816 Gly Ile Ile GlyAsn Gly Ala Gly Leu Val Met Ser Thr Leu Asp Ile 260 265 270 gtg gct gcagct ggt gaa cgc cat ggt ggg cag cgc ccc gcg aac ttc 864 Val Ala Ala AlaGly Glu Arg His Gly Gly Gln Arg Pro Ala Asn Phe 275 280 285 cta gac attggt ggc gga gca tca gct gaa tcg atg gct gct ggt ctc 912 Leu Asp Ile GlyGly Gly Ala Ser Ala Glu Ser Met Ala Ala Gly Leu 290 295 300 gat gtg atcctt ggg gat agc cag gta cgc agt gtg ttt gtg aat gtg 960 Asp Val Ile LeuGly Asp Ser Gln Val Arg Ser Val Phe Val Asn Val 305 310 315 320 ttt ggtggc atc acc gcg tgt gat gtg gtg gca aag gga atc gtt gga 1008 Phe Gly GlyIle Thr Ala Cys Asp Val Val Ala Lys Gly Ile Val Gly 325 330 335 gct ttggat gtg ctc ggc gat caa gca acg aag cct ctt gtg gtg cgc 1056 Ala Leu AspVal Leu Gly Asp Gln Ala Thr Lys Pro Leu Val Val Arg 340 345 350 ctt gatggc aac aac gtg gtg gaa ggc aga cga atc ctc gcg gaa tat 1104 Leu Asp GlyAsn Asn Val Val Glu Gly Arg Arg Ile Leu Ala Glu Tyr 355 360 365 aac caccct ttg gtc acc gtt gtg gag ggt atg gat gca gcg gct gat 1152 Asn His ProLeu Val Thr Val Val Glu Gly Met Asp Ala Ala Ala Asp 370 375 380 cac gctgcc cat ttg gcc aat ctt gcc cag cac ggc cag ttc gca acc 1200 His Ala AlaHis Leu Ala Asn Leu Ala Gln His Gly Gln Phe Ala Thr 385 390 395 400 gctaat 1206 Ala Asn 5 402 PRT Corynebacterium glutamicum 5 Met Glu Leu AlaVal Asp Leu Phe Glu Tyr Gln Ala Arg Asp Leu Phe 1 5 10 15 Glu Thr HisGly Val Ser Val Leu Lys Gly Ile Val Ala Ser Thr Pro 20 25 30 Glu Ala AlaArg Lys Ala Ala Glu Glu Ile Gly Glu Leu Thr Val Val 35 40 45 Lys Ala GlnVal Lys Val Gly Gly Arg Gly Lys Ala Gly Gly Val Arg 50 55 60 Val Ala ProThr Ser Ala Gln Ala Phe Asp Ala Ala Asp Ala Ile Leu 65 70 75 80 Gly MetAsp Ile Lys Gly His Thr Val Asn Gln Val Met Val Ala Gln 85 90 95 Gly AlaAsp Ile Ala Glu Glu Tyr Tyr Phe Ser Ile Leu Leu Asp Arg 100 105 110 AlaAsn Arg Ser Tyr Leu Ala Met Cys Ser Val Glu Gly Gly Met Glu 115 120 125Ile Glu Ile Leu Ala Lys Glu Lys Pro Glu Ala Leu Ala Lys Val Glu 130 135140 Val Asp Pro Leu Thr Gly Ile Asp Glu Asp Lys Ala Arg Glu Ile Val 145150 155 160 Thr Ala Ala Gly Phe Glu Thr Glu Val Thr Glu Lys Val Ile ProVal 165 170 175 Leu Ile Lys Ile Trp Gln Val Tyr Tyr Glu Glu Glu Ala ThrLeu Val 180 185 190 Glu Val Asn Pro Leu Val Leu Thr Asp Asp Gly Asp ValIle Ala Leu 195 200 205 Asp Gly Lys Ile Thr Leu Asp Asp Asn Ala Asp PheArg His Asp Asn 210 215 220 Arg Gly Ala Leu Ala Glu Ser Ala Gly Gly LeuAsp Ile Leu Glu Leu 225 230 235 240 Lys Ala Lys Lys Asn Asp Leu Asn TyrVal Lys Leu Asp Gly Ser Val 245 250 255 Gly Ile Ile Gly Asn Gly Ala GlyLeu Val Met Ser Thr Leu Asp Ile 260 265 270 Val Ala Ala Ala Gly Glu ArgHis Gly Gly Gln Arg Pro Ala Asn Phe 275 280 285 Leu Asp Ile Gly Gly GlyAla Ser Ala Glu Ser Met Ala Ala Gly Leu 290 295 300 Asp Val Ile Leu GlyAsp Ser Gln Val Arg Ser Val Phe Val Asn Val 305 310 315 320 Phe Gly GlyIle Thr Ala Cys Asp Val Val Ala Lys Gly Ile Val Gly 325 330 335 Ala LeuAsp Val Leu Gly Asp Gln Ala Thr Lys Pro Leu Val Val Arg 340 345 350 LeuAsp Gly Asn Asn Val Val Glu Gly Arg Arg Ile Leu Ala Glu Tyr 355 360 365Asn His Pro Leu Val Thr Val Val Glu Gly Met Asp Ala Ala Ala Asp 370 375380 His Ala Ala His Leu Ala Asn Leu Ala Gln His Gly Gln Phe Ala Thr 385390 395 400 Ala Asn

What is claimed is:
 1. Isolated polynucleotide containing apolynucleotide sequence coding for the sucC- and/or sucD-gene, selectedfrom the group comprising a) Polynucleotide that is at least 70%identical to a polynucleotide coding for a polypeptide that contains theamino acid sequence of SEQ ID No. 2, b) Polynucleotide that is at least70% identical to a polynucleotide coding for a polypeptide that containsthe amino acid sequence of SEQ ID No. 3, c) Polynucleotide coding for apolypeptide that contains an amino acid sequence that is at least 70%identical to that of the amino acid sequence of SEQ ID No. 2, d)Polynucleotide coding for a polypeptide that contains an amino acidsequence that is at least 70% identical to that of the amino acidsequence of SEQ ID No. 3, e) Polynucleotide that is complementary to thepolynucleotides of a), b), c) or d), and f) Polynucleotide containing atleast 15 successive nucleotides of the polynucleotide sequences of a),b), c), d) or e), the polypeptide preferably having the activity ofsuccinyl-CoA synthetase.
 2. Polynucleotides according to claim 1,wherein the polynucleotide is a preferably recombinant DNA that isreplicable in coryneform bacteria.
 3. Polynucleotides according to claim1, wherein the polynucleotide is a RNA.
 4. Polynucleotides according toclaim 2, containing the nucleic acid sequence as shown in SEQ ID NO 1.5. Replicable DNA according to claim 2, containing (i) the nucleotidesequence shown in SEQ ID NO 1, or (ii) at least one sequence thatcorresponds to the sequences (i) within the region of degeneration ofthe genetic code, or (iii) at least one sequence that hybridizes withthe sequences that are complementary to the sequences (i) or (ii), andoptionally (iv) functionally neutral sense mutations in (i). 6.Replicable. DNA according to claim 5, wherein the hybridization iscarried out under a stringency corresponding to at most 2×SSC. 7.Polynucleotide sequence according to claim 1 that codes for apolypeptide that contains the amino acid sequence shown in SEQ ID No. 2.8. Coryneform bacteria in which the sucC- and/or sucD-gene is/areattenuated.
 9. Process for producing L-amino acids, in particularL-lysine and/or L-glutamate, wherein the following steps are carriedout: a) fermentation of the bacteria producing the desired L-amino acid,in which first of all the sucC- and/or sucD-gene or nucleotide sequencescoding therefor are attenuated, in particular switched off; b)enrichment of the L-amino acid in the medium or in the bacterial cells,and c) isolation of the L-amino acid.
 10. Process according to claim 9,wherein bacteria are used in which in addition further genes of thebiosynthesis pathway of the desired L-amino acid are enhanced. 11.Process according to claim 9, wherein bacteria are used in which themetabolic pathways that reduce the formation of the desired L-amino acidare at least partially switched off.
 12. Process according to claim 9,wherein the expression of the polynucleotide(s) that codes for the sucC-and/or sucD-genes is attenuated, in particular is switched off. 13.Process according to claim 9, wherein the catalytic properties of thepolypeptide (enzyme protein) for which the polynucleotides sucC and sucDcode are reduced.
 14. Process according to claim 9, wherein for theproduction of L-amino acids microorganisms are fermented in which at thesame time one or more of the genes selected from the following groupis/are enhanced and/or overexpressed: 14.1 the dapA-gene coding fordihydrodipicolinate synthase, 14.2 the pyc-gene coding for pyruvatecarboxylase, 14.3 the gap-gene coding for glyceraldehyde-3-phosphatedehydrogenase, 14.4 the gene tpi coding for triose phosphate isomerase,14.5 the gene pgk coding for 3-phosphoglycerate kinase, 14.6 themqo-gene coding for malate:quinone oxidoreductase, 14.7 the lysE-genecoding for L-lysine export, 14.8 the gene lysC coding for a feedbackresistant aspartate kinase, 14.9 the gene zwa1 coding for theZwa1-protein.
 15. Process according to claim 9, wherein for theproduction of L-amino acids coryneform microorganisms are fermented, inwhich at the same time one or more of the genes selected from thefollowing group is/are attenuated: 15.1 the gene pck coding forphosphoenol pyruvate carboxykinase, 15.2 the gene pgi coding forglucose-6-phosphate isomerase, 15.3 the gene poxB coding forpyruvate-oxidase, 15.4 the gene zwa2 coding for the Zwa2-Protein. 16.Coryneform bacteria containing a vector that carries parts of thepolynucleotide according to claim 1, but at least 15 successivenucleotides of the claimed sequence.
 17. DNA derived from coryneformbacteria that code for SucC proteins, whose amino acid sequence (SEQ IDNo. 2) contains one or more replacements selected from the group:replacement at position 22 by any other proteinogenic amino acid exceptL-proline, replacement at position 44 by any other proteinogenic aminoacid except glycine, and replacement at position 170 by any otherproteinogenic amino acid except L-alanine.
 18. DNA according to claim17, wherein this codes for SucC proteins whose amino acid sequencescontain one or more replacements selected from the group: L-proline atposition 22 by L-serine, glycine at position 44 by L-glutamic acid, andL-alanine at position 170 by L-threonine.
 19. DNA according to claim 18,wherein this codes for a SucC protein whose amino acid sequence containsL-serine at position 22, L-glutamic acid at position 44, and L-threonineat position 170, as illustrated in SEQ ID No.5.
 20. DNA according toclaim 17, wherein this contains the nucleobase thymine at position 64,the nucleobase adenine at position 131, and the nucleobase adenine atposition 508, as illustrated in SEQ ID No
 4. 21. Coryneforme bacteriathat contain a DNA according to claim 17, 18, 19 or
 20. 22. Integrationvector pCRBluntsucCint, that 22.1 carries a 0.55 kb long internalfragment of the sucC-gene, 22.2 whose restriction map is reproduced inFIG. 1, and 22.3 that in the E. coli strain TOP10/pCRBluntsucCint hasbeen filed under No. DSM 13750 at the German Collection forMicroorganisms and Cell Cultures.
 23. Plasmid vector pK18mobsacBsucDdelthat 23.1 carries a sucD-gene containing a deletion, 23.2 whoserestriction map is reproduced in FIG. 2, and 23.3 that in the E. colistrain DH5αmcr/pK18mobsacBsucDdel has been filed under No. DSM 13749 atthe German Collection for Microorganisms and Cell Cultures.
 24. Processaccording to one or more of the preceding claims, wherein microorganismsof the type Corynebacterium glutamicum are used.
 25. Process fordetecting RNA, cDNA and DNA in order to isolate nucleic acids and/orpolynucleotides or genes that code for succinyl-CoA synthase or thathave a high degree of similarity to the sequence of the sucC- and/orsucD-gene, wherein the polynucleotide containing the polynucleotidesequences according to claims 1, 2, 3 or 4, is used as hybridizationprobe.